eNOS, as a synthesizer of nitric oxide (NO), plays an important role in the regulation of vascular tone, platelet and leukocyte adhesion to the endothelium, endothelin-1 generation, vascular smooth muscle proliferation, and vascular lesion formation. Recently, it has been demonstrated that eNOS directly binds to the 90 kDa heat shock proteins, Hsp90, however, the interacting domains in the two proteins have not been identified. Increased interaction of Hsp90 with eNOS has been demonstrated with a variety of treatments and is associated with increased eNOS activity, which can be blocked by the specific inhibitor of Hsp90, geldanamycin. The mechanism for Hsp90 activation of eNOS is unknown. Due to its role as a molecular chaperone, Hsp90 may stabilize and prevent the aggregation of partially folded or dimeric forms of eNOS, thereby increasing the overall activity. In addition, Hsp90 may alter the three dimensional structure of eNOS and affect binding of important co-factors such as calmodulin (CaM). Therefore, the goals of this project are two-fold, to map the interacting domains between Hsp90 and eNOS and to examine potential mechanisms by which Hsp90 upregulates eNOS activity.